The export of messenger (m)RNA from the nucleus to the cytoplasm is an essential step in the eukaryotic gene expression pathway and impacts all aspects of cell physiology. The goal of this project is to elucidate the precise mechanism of human mRNA export by deciphering the function of two essential proteins, human (h) GlelB and hDbp5. Using the budding yeast (y) S. cerevisiae model, our laboratory recently found that yGlel, bound to inositol hexakisphosphate (IP6), is required to maximally stimulate the RNA-dependent ATPase activity of yDbpS. Conversion of yDbp5 to the ADP-bound form then triggers changes in the protein composition of the exported messenger ribonucleoprotein particle (mRNP). We hypothesize that the mechanism of export is conserved in human cells, and perturbed in a lethal human motoneuron degenerative disease. To analyze the process in human cells and test this hypothesis, two aims are proposed. In aim one, the biochemical properties of recombinant purified hGlel B and hDbp5 will be analyzed. This work will utilize a battery of assays that are well established in our laboratory. In vitro binding assays will be performed to test for IP6 interaction with hGlel B. To test for activation, ATPase assays will be performed with hDbp5, hGlel B and IPs. As hDbp5 potentially acts as both an RNA helicase and an RNP remodeler, direct assays will be conducted for the role of hGlel B and IP6 in these functions. In vitro protein- protein interactions with recombinant hGlel B and hDbpS will be conducted, as well as in vivo co- immunoprecipitation studies from human tissue culture cells. Results of these assays will provide evidence for the specific functions of hGlel B, IP6, and hDbpS in mRNA export, and potentially identify requirements for other novel cellular co-factors. In aim two, the mechanism by which the hGlel B-Finmajor mutation is perturbed will be investigated. This mutant allele was recently reported as the causal link in LCCS1 (lethal congenital contractile syndrome 1). Recombinant purified hGlel B-Finmajor protein will be assayed for IP6 binding, hDbpS activation and binding. Transient expression experiments in human tissue culture cells will be used to compare the subcellular localizations of wild-type and hGlel B-Finmajor proteins. Using siRNA knockdown strategies, wild-type hGlel will be replaced by expression of hGlel B-Finmaj0r and in situ assays for mRNA export function will be conducted. These studies will give insight into the disease mechanism. Public Health Relevance: mRNA export is a basic cellular process that is essential to the viability of eukaryotic cells. Dysregulation of this process is implicated in viral infection, cancer processes, and now potentially in neuronal development. Understanding the normal export mechanism will allow perturbations in such disease processes to be detected and targeted for treatment.